JoVE General
Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq
Department of Biology, New Mexico State University
A ribosomal RNA (rRNA) depletion protocol was developed to enrich messenger RNA (mRNA) for RNA-seq of the mosquito gut metatranscriptome. Sample specific rRNA probes, which were used to remove rRNA via subtraction, were created from the mosquito and its gut microbes. Performance of the protocol can result in the removal of approximately 90-99% of rRNA.
JoVE General
Bromodeoxyuridine (BrdU) Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton
1Biological Sciences, Kent State University, 2Marine Sciences, University of Georgia (UGA)
Environmental bacterioplankton are incubated with a model dissolved organic carbon (DOC) compound and a DNA labeling reagent, bromodeoxyuridine (BrdU). Afterward, DOC-degrading cells are separated from the bulk community based on their elevated BrdU incorporation using fluorescence activated cell sorting (FACS). These cells are then identified by subsequent molecular analyses.
JoVE General
DNA Stable-Isotope Probing (DNA-SIP)
Department of Biology, University of Waterloo
DNA stable-isotope probing is a cultivation-independent method to identify and characterize active communities of microorganisms that are capable of utilizing specific substrates. Assimilation of substrate enriched in heavy isotope leads to incorporation of labelled atoms into microbial biomass. Density gradient ultracentrifugation retrieves labelled DNA for downstream molecular analyses.
JoVE General
Extraction of High Molecular Weight DNA from Microbial Mats
We provide an improved protocol for extracting high molecular weight DNA from hypersaline microbial mats. Microbial cells are separated from the mat matrix prior to DNA extraction and purification. This enhances the concentrations, quality, and size of the DNA. The protocol may be used for other refractory samples.
JoVE General
Concentration of Metabolites from Low-density Planktonic Communities for Environmental Metabolomics using Nuclear Magnetic Resonance Spectroscopy
1Biosphere Oriented Biology Research Unit, RIKEN Advanced Science Institute, 2Graduate School of Nanobioscience, Yokohama City University, 3Advanced NMR Metabomics Research Team, RIKEN Plant Science Center, 4Graduate School of Bioagricultural Science, Nagoya University
A method for metabolite extraction from microbial planktonic communities is presented. Whole community sampling is achieved by filtration onto specially prepared filters. After lyophilization, aqueous-soluble metabolites are extracted. This approach allows for application of environmental metabolomics to trans-omics investigations of natural or experimental microbial communities.
JoVE Bioengineering
Bacterial Detection & Identification Using Electrochemical Sensors
1Research Service, Veterans Affairs Greater Los Angeles Healthcare System, 2Department of Urology, The David Geffen School of Medicine, University of California, Los Angeles, 3GeneFluidics, 4Division of Infectious Diseases, Veterans Affairs Greater Los Angeles Healthcare System, 5Department of Microbiology, Immunology & Molecular Genetics, University of California, Los Angeles
We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.
JoVE General
Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)
1Department of Biochemistry / Analytics, caprotec bioanalytics GmbH, 2Institute of Organic Chemistry, RWTH Aachen University
Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.
JoVE General
Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment
1School of Earth Sciences, The Ohio State University, 2School of Environment & Natural Resources, The Ohio State University, 3Institute of Geology and Geophysics, Chinese Academy of Sciences
We demonstrate a method to collect magnetotactic bacteria (MTB) that can be applied to natural waters. MTB can be isolated and enriched from sediment samples using a relatively simple setup that takes advantage of the bacteria's natural magnetism. Isolated MTB can then be examined in detail using both light and electron microscopy.
JoVE Clinical and Translational Medicine
Sequencing of Bacterial Microflora in Peripheral Blood: our Experience with HIV-infected Patients
Our experiment will show how to perform a sequencing analysis of bacterial species translocating in peripheral blood of HIV positive patients.
JoVE Immunology and Infection
One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures
1Department of Medical Microbiology, Maastricht University Medical Center, 2Department of Internal Medicine, Erasmus Medical Center
The design of a straightforward one-day workflow scheme for bacterial pathogen diagnostics enables the rapid recognition of bloodstream infections. The inclusion of eight clinically relevant bacterial targets and their antibiotic resistance profiles offers the clinician an initial insight on the same day, which can lead to more adequate therapy.
JoVE Bioengineering
On-chip Isotachophoresis for Separation of Ions and Purification of Nucleic Acids
Mechanical Engineering, Stanford University
Isotachophoresis (ITP) is a robust electrokinetic separation and preconcentration technique with applications ranging from toxin detection to sample preparation. We review the physical principles of ITP and the methodology of applying this technique to two specific example applications: separation and detection of small molecules and purification of nucleic acids from cell culture lysate.
JoVE General
Extracting DNA from the Gut Microbes of the Termite (Zootermopsis Angusticollis) and Visualizing Gut Microbes
Department of Environmental Science and Engineering, California Institute of Technology - Caltech
This video illustrates the technique for extracting DNA from the species of microbes resident in the termite hindgut. The preparation of a wet mount slide, which is useful for visualizing the gut microbial community is also illustrated, and a tour through the species-rich gut environment is given.
JoVE Immunology and Infection
RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract
1Department of Pediatrics, University of Texas Southwestern Medical Center, 2Channing Laboratory, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, 3Department of Pediatrics and Microbiology, University of Texas Southwestern Medical Center
A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes.
JoVE Application Notes
LookOut Mycoplasma Elimination Kit - ADVERTISEMENT
Product Management, Sigma-Aldrich
The LookOut Mycoplasma elimination kit combines biological agents that reliably and completely eliminate mycoplasma contamination with minimal cytotoxic effect on cells.
JoVE Editorial
JoVE 4th Issue
JoVE Immunology and Infection
RNA Interference in Ticks
1Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University, 2(CSIC-UCLM-JCCM), Instituto de Investigación en Recursos Cinegéticos IREC
A method for RNA interference (RNAi) by injection of dsRNA into unfed ticks is described. RNAi is the most widely used gene-silencing technique in ticks where the use of other methods of genetic manipulation has been limited.
JoVE General
Cryosectioning Yeast Communities for Examining Fluorescence Patterns
Division of Basic Sciences, Fred Hutchinson Cancer Research Center
We present a protocol for freezing and cryosectioning yeast communities to observe internal patterns of fluorescent cells. The method relies on methanol-fixing and OCT-embedding to preserve the spatial distribution of cells without inactivating fluorescent proteins within a community.
JoVE General
Analyzing Gene Expression from Marine Microbial Communities using Environmental Transcriptomics
Department of Marine Sciences, University of Georgia (UGA)
We present a method for generating cDNA from environmental mRNA. In general, total RNA is first collected from the environment, rRNA is selectively removed, mRNA is selectively amplified, and cDNA synthesized from the enriched mRNA pool is sequenced. Recovered sequences can be annotated using standard bioinformatics techniques to identify the expressed genes.
JoVE Immunology and Infection
Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry
Institute for Molecular Infection Biology (IMIB), University of Würzburg
Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.
JoVE General
RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells
Children's Mercy Hospital and Clinics, School of Medicine, University of Missouri-Kansas City
This protocol presents a complete and detailed procedure to apply RNA-seq, a powerful next-generation DNA sequencing technology, to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is generalizable to various cells or tissues affected by different reagents or disease states.
JoVE Clinical and Translational Medicine
Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy
1Department of Orthodontics and Maxillofacial Orthopedics, Medical University of Graz, 2Institute of Hygiene, Microbiology and Environmental Medicine, Medical University of Graz, 3Department of Prosthodontics, Restorative Dentistry, Periodontology and Implantology, Medical University of Graz, 4Institute of Plant Sciences, Karl-Franzens-University Graz
We present a protocol for structural and compositional analysis of natural oral biofilm from orthodontic appliances with in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). Oral biofilm samples were collected from palatal expanders, scraping acrylic-resin flakes off their surface and referring them for molecular processing.
JoVE General
Multiplex PCR and Reverse Line Blot Hybridization Assay (mPCR/RLB)
Centre for Infectious Diseases and Microbiology, University of Sydney
An inexpensive, high throughput method for simultaneous detection of up to 43 molecular targets is described. Applications of mPCR/RLB include microbial typing and detection of multiple pathogens from clinical samples.
JoVE Immunology and Infection
Estimating Virus Production Rates in Aquatic Systems
Department of Microbiology, University of Tennessee
The turnover rate of viruses in marine and freshwater systems can be estimated by a reduction and reoccurrence technique. The data allow researchers to infer rates of virus-mediated microbial mortality in aquatic systems.
JoVE General
Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses
1Department of Biological Sciences, University of Alabama Huntsville, 2Department of Biology, Stanford University
A major impediment to biochemical analyses of ribosomes containing nascent peptidyl-tRNAs has been the presence of other ribosomes in the same samples, ribosomes not involved in the translation of the specific mRNA sequence being analyzed. We developed a simple methodology to purify, exclusively, the ribosomes containing the nascent peptidyl-tRNA of interest.
JoVE General
Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks
1Molecular and Microbiology Department and Center for the Study of Genomics in Liver Diseases, George Mason University, 2Translational Research Institute, Inova Health System, 3Research Center for Medical Genetics RAMS
Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.
JoVE Clinical and Translational Medicine
Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract
1St. Erik's Eye Hospital, Karolinska Institutet, 2Gullstrand lab, Section for Ophthalmology, Department of Neuroscience, Uppsala University
Cataract is the leading cause of blindness in the world. Solar ultraviolet radiation (UVR) is the main risk factor for cataract development. An animal model of far UVR-B induced cataract was developed. In this article we describe methods for investigation of cataract formation: exposure to UVR, quantitative RT-PCR and immunohistochemistry.
JoVE General
Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency
Gene Expression Division, Bio-Rad Laboratories, Inc.
This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.
JoVE Immunology and Infection
Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography
1Department of Microbial and Environmental Genomics, The J. Craig Venter Institute, 2Department of Synthetic Biology and Bioenergy, The J. Craig Venter Institute
We describe an efficient method to separate single-stranded DNA, double-stranded DNA and RNA molecules from environmental viral communities. Nucleic acids are fractionated using hydroxyapatite chromatography with increasing concentrations of phosphate-containing buffers. This method permits the isolation of all viral nucleic acid types from environmental samples.
JoVE Neuroscience
Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection
Department of Biochemistry and Molecular Biology, Drexel University College of Medicine
In this video article we describe the use of a new ex vivo model of acute herpes simplex virus type I corneal epithelial infection.
JoVE General
RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis
School of Health Sciences, Purdue University
The isolation of high quality, intact RNA is an essential step in many laboratory protocols. Here, we demonstrate RNA extraction from whole zebrafish embryos and cDNA synthesis for subsequent application in various experimental procedures including gene expression microarray analysis.
JoVE General
The ITS2 Database
1Department of Bioinformatics, Biocenter, University of Würzburg, 2Institute of Pharmacy and Food Chemistry, University of Würzburg
The ITS2 Database is a workbench for phylogenetic inference simultaneously considering sequence and secondary structure of the internal transcribed spacer 2. This includes data collection with accurate annotation, structure prediction, multiple sequence-structure alignment and fast tree calculation. In a nutshell, this workbench simplifies first phylogenetic analyses to a few clicks.
JoVE General
Primer Extension Capture: Targeted Sequence Retrieval from Heavily Degraded DNA Sources
Max-Planck Institute for Evolutionary Anthropology, Leipzig
We present a method of targeted ancient DNA sequence retrieval, which we used to reconstruct the complete mitochondrial genomes of five Neandertal individuals. Comparison of these sequences with present day humans suggests that Neandertals had a long term low effective population size.
JoVE General
Isolation and Characterization of RNA-Containing Exosomes
This paper demonstrates methods for the isolation, purification and detection of exosomes, as well as techniques for analysis of their molecular content. These methods are adaptable for exosome isolation from both cell culture media and biological fluids, and can beyond analysis of molecular content also be useful in functional studies.
JoVE General
The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation
Gene Expression Division, Bio-Rad Laboratories, Inc
This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.
JoVE General
Quantitative Real-Time PCR using the Thermo Scientific Solaris qPCR Assay
Thermo Scientific Solaris qPCR Products
The Solaris qPCR Gene Expression Assays are novel pre-designed qPCR primer/probe combinations designed to simplify the qPCR process without sacrificing the specificity and robustness of the assay.
JoVE General
Gene Transfer into Older Chicken Embryos by ex ovo Electroporation
1Albrecht-Kossel-Institute for Neuroregeneration, School of Medicine University of Rostock, 2Institute of Anatomy I, School of Medicine University of Jena
A method of gene transfer into chicken embryos at later incubation stages (older than Hamburger and Hamilton stage (HH) 22) is described. This method overcomes disadvantages of in ovo electroporation applied to older chicken embryos and is a useful technique to study gene function and regulation at older developmental stages.
JoVE General
Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer's Vesicle
1Department of Cell and Developmental Biology, State University of New York, Upstate Medical University, 2Department of Neurobiology and Anatomy, Eccles Institute of Human Genetics, University of Utah
Cilia-generated fluid flow in Kupffer’s Vesicle (KV) controls left-right patterning of the zebrafish embryo. Here, we describe a technique to modulate gene function specifically in KV cells. In addition, we show how to deliver fluorescent beads into KV to visualize fluid flow.
JoVE General
Purification of Transcripts and Metabolites from Drosophila Heads
1Department of Neurology, McKnight Brain Institute, University of Florida, 2Department of Entomology and Nematology, University of Florida, 3Genetics Institute, Department of Molecular Genetics and Microbiology, University of Florida, 4McKnight Brain Institute, Department of Neuroscience, Genetics Institute, Center for Translational Research on Neurodegenerative Diseases, and Center for Movement Disorders and Neurorestoration, University of Florida
We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.
JoVE General
Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes
Department of Biological Sciences, University of Alabama
This video demonstrates how to use C. elegans to assess dopaminergic neuron neurodegeneration as a model for Parkinson's disease. Furthermore, genetic screens are used to identify factors that either enhance degeneration or are neuroprotective.
JoVE General
Preparation of Gene Gun Bullets and Biolistic Transfection of Neurons in Slice Culture
Center for Neuroscience, University of California, Davis
We describe a method for preparing DNA coated gold bullets and demonstrate the use of such bullets to biolistically transfect neurons in cultured hippocampal slices.
JoVE Clinical and Translational Medicine
Ex Vivo Culture of Patient Tissue & Examination of Gene Delivery
1Cork Cancer Research Centre, Mercy University Hospital and Leslie C. Quick Jnr. Laboratory, University College Cork, 2Department of Computer Science, University College Cork
This article describes the culture of patient tissue slices for gene delivery studies and subsequent analysis of gene expression using IVIS bioluminescence imaging.
JoVE General
Annotation of Plant Gene Function via Combined Genomics, Metabolomics and Informatics
Molekulare Pflanzenphysiologie, Max-Planck-Institut
Combination of genomics, co-expression gene analysis and the identification of target compounds via metabolism give gene functional annotation.
JoVE Immunology and Infection
DNA Fingerprinting of Mycobacterium leprae Strains Using Variable Number Tandem Repeat (VNTR) - Fragment Length Analysis (FLA)
Department of Microbiology, Immunology and Pathology, Colorado State University
Leprosy, caused by Mycobacterium leprae, is still endemic in many places. In order to learn about the spread and mode of transmission of leprosy, it is important to determine which strain of M. leprae has infected a patient. Variable numbers of tandem repeats (VNTR) typing is one such method.
JoVE Clinical and Translational Medicine
Movement Retraining using Real-time Feedback of Performance
Department of Physical Therapy, University of British Columbia
Retraining abnormal movement patterns following injury or disease is a key component of physical rehabilitation. Recent advances in technology have permitted accurate assessment of movement during a variety of tasks, with near instantaneous quantification of results. This provides new opportunities for modification of faulty movement patterns in real time.
JoVE Bioengineering
Continuously-stirred Anaerobic Digester to Convert Organic Wastes into Biogas: System Setup and Basic Operation
Department of Biological and Environmental Engineering, Cornell University
Laboratory-scale anaerobic digesters allow scientists to research new ways of optimizing existing applications of anaerobic biotechnology and to evaluate the methane producing potential of various organic wastes. This article introduces a generalized model for the construction, inoculation, operation, and monitoring of a laboratory-scale continuously stirred anaerobic digester.
JoVE General
Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans
1Department of Science, Borough of Manhattan Community College, City Universtiy of New York (CUNY), 2Department of Biology, Queens College, The City University of New York (CUNY), 3Biochemistry Program, The Graduate Center, Queens College, The City University of New York (CUNY)
We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-β signaling.
JoVE Neuroscience
Micromanipulation of Gene Expression in the Adult Zebrafish Brain Using Cerebroventricular Microinjection of Morpholino Oligonucleotides
In this article, we demonstrate a method for manipulation of gene expression in the ventricular cells of the adult zebrafish telencephalon using antisense morpholino oligonucleotides. We present this method as an efficient and quick protocol that can be used for functional studies in the adult vertebrate brain.
JoVE General
Yeast Colony Embedding Method
School of Biological Sciences, University of Missouri - Kansas City
A method for embedding yeast colonies allowing sectioning for light and electron microscopy. This protocol allows determination of the distribution of sporulated cells and pseudohyphal cells within colonies providing a new tool toward understanding the organization of cell types within a fungal community.
JoVE General
Generation of Stable Transgenic C. elegans Using Microinjection
Department of Biological Sciences, University of Alabama
This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.
JoVE General
Extraction of High Molecular Weight Genomic DNA from Soils and Sediments
Department of Microbiology and Immunology, University of British Columbia - UBC
A methodology to isolate high molecular weight and high quality genomic DNA from soil microbial community is described.
More Results...
